The lncRNA Sweetheart regulates compensatory cardiac hypertrophy after myocardial injury in murine males.

After myocardial infarction in the adult heart the remaining, non-infarcted tissue adapts to compensate the loss of functional tissue. This adaptation requires changes in gene expression networks, which are mostly controlled by transcription regulating proteins. Long non-coding transcripts (lncRNAs) are taking part in fine-tuning such gene programs. We describe and characterize the cardiomyocyte specific lncRNA Sweetheart RNA (Swhtr), an approximately 10 kb long transcript divergently expressed from the cardiac core transcription factor coding gene Nkx2-5. We show that Swhtr is dispensable for normal heart development and function but becomes essential for the tissue adaptation process after myocardial infarction in murine males. Re-expressing Swhtr from an exogenous locus rescues the Swhtr null phenotype. Genes that depend on Swhtr after cardiac stress are significantly occupied and therefore most likely regulated by NKX2-5. The Swhtr transcript interacts with NKX2-5 and disperses upon hypoxic stress in cardiomyocytes, indicating an auxiliary role of Swhtr for NKX2-5 function in tissue adaptation after myocardial injury.

Mitochondrial-cell cycle cross-talk drives endoreplication in heart disease.

Endoreplication, duplication of the nuclear genome without cell division, occurs in disease to drive morphologic growth, cell fate, and function. Despite its criticality, the metabolic underpinnings of disease-induced endoreplication and its link to morphologic growth are unknown. Heart disease is characterized by endoreplication preceding cardiac hypertrophy. We identify ATP synthase as a central control node and determinant of cardiac endoreplication and hypertrophy by rechanneling free mitochondrial ADP to methylenetetrahydrofolate dehydrogenase 1 L (MTHFD1L), a mitochondrial localized rate-limiting enzyme of formate and de novo nucleotide biosynthesis. Concomitant activation of the adenosine monophosphate­activated protein kinase (AMPK)­retinoblastoma protein (Rb)-E2F axis co-opts metabolic products of MTHFD1L function to support DNA endoreplication and pathologic growth. Gain- and loss-of-function studies in genetic and surgical mouse heart disease models and correlation in individuals confirm direct coupling of deregulated energetics with endoreplication and pathologic overgrowth. Together, we identify cardiometabolic endoreplication as a hitherto unknown mechanism dictating pathologic growth progression in the failing myocardium.

Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction.

Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

Analysis of Cell Type-Specific Effects of MicroRNA-92a Provides Novel Insights Into Target Regulation and Mechanism of Action.

BACKGROUND: MicroRNAs (miRs) regulate nearly all biological pathways. Because the dysregulation of miRs can lead to disease progression, they are being explored as novel therapeutic targets. However, the cell type-specific effects of miRs in the heart are poorly understood. Thus, we assessed miR target regulation using miR-92a-3p as an example. Inhibition of miR-92a is known to improve endothelial cell function and recovery after acute myocardial infarction. METHODS: miR-92a-3p was inhibited by locked nucleic acid (LNA)-based antimiR (LNA-92a) in mice after myocardial infarction. Expression of regulated genes was evaluated 3 days after myocardial infarction by RNA sequencing of isolated endothelial cells, cardiomyocytes, fibroblasts, and CD45+ hematopoietic cells. RESULTS: LNA-92a depleted miR-92a-3p expression in all cell types and derepressed predicted miR-92a-3p targets in a cell type-specific manner. RNAseq showed endothelial cell-specific regulation of autophagy-related genes. Imaging confirmed increased endothelial cell autophagy in LNA-92a treated relative to control animals. In vitro inhibition of miR-92a-3p augmented EC autophagy, derepressed autophagy-related gene 4a, and increased luciferase activity in autophagy-related gene 4a 3'UTR containing reporters, whereas miR-92a-3p overexpression had the opposite effect. In cardiomyocytes, LNA-92a derepressed metabolism-related genes, notably, the high-density lipoprotein transporter Abca8b. LNA-92a further increased fatty acid uptake and mitochondrial function in cardiomyocytes in vitro. CONCLUSIONS: Our data show that miRs have cell type-specific effects in vivo. Analysis of miR targets in cell subsets disclosed a novel function of miR-92a-3p in endothelial cell autophagy and cardiomyocyte metabolism. Because autophagy is upregulated during ischemia to supply nutrients and cardiomyocyte metabolic-switching improves available substrate utilization, these prosurvival mechanisms may diminish tissue damage.

Switch in Laminin ß2 to Laminin ß1 Isoforms During Aging Controls Endothelial Cell Functions-Brief Report.

OBJECTIVE: Endothelial cells play important roles in tissue homeostasis and vascularization, a function that is impaired by aging. Here, we aim to decipher the role of the microenvironment underlying the impairment of endothelial cell functions by aging. APPROACH AND RESULTS: RNA sequencing of isolated cardiac endothelial cells derived from young and 18-month-old mouse hearts revealed that aging affects the endothelial expression of genes encoding extracellular matrix proteins, specifically the laminin ß1 (Lamb1) and laminin ß2 (Lamb2) chains. Whereas Lamb1 was upregulated, Lamb2 was decreased in endothelial cells in old mice compared with young controls. A similar change in expression patterns was observed after induction of acute myocardial infarction. Mimicking aging and injury conditions by plating endothelial cells on laminin ß1-containing laminin 411 matrix impaired endothelial cell adhesion, migration, and tube formation and augmented endothelial-to-mesenchymal transition and endothelial detachment compared with laminin 421, which contains the laminin ß2 chain. Because laminins can signal via integrin receptors, we determined the activation of ITGB1 (integrin ß1). Laminin 421 coating induced a higher activation of ITGB1 compared with laminin 411. siRNA-mediated silencing of ITGB1 reduced laminin ß2-dependent adhesion, suggesting that laminin ß2 more efficiently activates ITGB1. CONCLUSIONS: Mimicking age-related modulation of laminin ß1 versus ß2 chain expression changes the functional properties and phenotype of endothelial cells. The dysregulation of the extracellular matrix during vascular aging may contribute to age-associated impairment of organ function and fibrosis.